Small Farm Holder Cropping Systems Influence Microbial Profiles in an Equatorial Rainforest Agroecosystem

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have a small communication that examined the bacteria/archaea and fungal communities from three habitats (forest, corn/soybean, and sugarcane) in Kenya. 

The manuscript is well written. However, the authors state they talk about fungi, but only focus on Ascomycota, which out explanation why the other fungal phylum are not discussed. Also, there are quite alot of bacterial and fungal species left out of the functional group discussion which they should mention why they were left out, and potentially how using only a small portion of the ASV would affect the interpretation of the results. Lastly, I would like the authors to add the averages of the soil characteristics, and not just discuss the p-values. All of these things would solidify the manuscript.

Comments for author File: Comments.pdf

Author Response

The authors have a small communication that examined the bacteria/archaea and fungal communities from three habitats (forest, corn/soybean, and sugarcane) in Kenya.

The manuscript is well written. However, the authors state they talk about fungi, but only focus on Ascomycota, which out explanation why the other fungal phylum are not discussed. Also, there are quite a lot of bacterial and fungal species left out of the functional group discussion which they should mention why they were left out, and potentially how using only a small portion of the ASV would affect the interpretation of the results. Lastly, I would like the authors to add the averages of the soil characteristics, and not just discuss the p-values. All of these things would solidify the manuscript.

Abstract

Lines 10-12, (“Metabarcoding of prokaryotic and fungal ribosomal DNA…”), the remaining discussion within this article is only on Ascomycota. Did the author find no other fungal phyla? This would be unlikely. If the authors are focusing on Ascomycota, then I would limit the terminology and state that somewhere.

Response: Yes, the primers used for fungi were specific to Ascomycota (indicated on lines 65-68), so other fungal phyla were not detected. This did limit the functional groups of fungi encountered but each site was sampled equally in this respect. We have revised the text in lines 17 and added new text in lines 112-122 to make it clear that we were focusing only on Ascomycota:

Of the 151 Ascomycota ASVs queried through FUNGuild, 59 taxa (39.1%) were assigned a functional trait, with most unassigned ASVs missing family, genus and species classification. It is likely that the naming conventions from the SILVA database (e.g. Cladosporium complex) were not directly matched within FUNGuild, and some others may be missing from the dataset (Furcaspora). Within the prokaryote dataset, there were 379 ASVs queried, and 59 were assigned a functional group, most classified to at least family. Unassigned taxa were those that were missing lower rank names, missing from the database, or remained unmatched due to SILVA naming (e.g., Candidatus Kroibacter, genus RB41, and others). There were a larger proportion of named prokaryotes (down to family or genus) that were not found in the FAPROTAX database (100 ASVs, 26.4%) than Ascomycota not found in FUNGuild (9 ASVs, 6.0%).

Methods

Lines 47-51, the results from this are missing within the manuscript. Please add.

Response: We have added an additional table (Table S1) in supplementary material with averages and standard deviation values. See lines 84-86:

In contrast, the two cropping systems occurred in a broad range of soil conditions—sandy to clay/silt rich, low to high acidity, low to high organic carbon and nitrogen (both N (%) and TN (Mg ha-1)) (Table 1; Table S1).

Raw data are available for download in the FigShare repository. Lines 77-79 address this further, as does the Data Availability statement at the bottom of the article.

Results

Lines 67-69, for this sentence, the authors should add the results from the chemical analyses, and add (Table X).

Response: See the new table (Table S1) in supplementary material indicated in the response above.

Table 1, Here the p-values have little meaning without raw data (averages and StDev).

Response: See the new table (Table S1) in supplementary material indicated in the response above.

Line 83, once again, the author mention fungi above, but limit the discussion to only Ascomycota. If the authors have found other fungal phylum, they need to include that or they need to state somewhere that they only focused on Ascomycota and why.

Response: The PCR primers used for fungi recover only Ascomycota (noted on lines 65-68); other fungal phyla were thus not detected.

Line 88, are the top most abundant within the forest all Ascomycota? Adding two additional sites with no ectomycorhizzal partners could skew the top 40 if lumping all sites together. 

Response: The possible presence in forest soils of ectomycorrhizal Basidiomycota that would not be present in maize-bean or sugarcane soils was not a biasing factor in the analyses, since the primers detected only Ascomycota; no ectomycorrhizal or ericoid mycorrhizal fungi were detected in any soils, as noted on lines 132-134:

All soils had primarily saprotrophic Ascomycota, with a smaller proportion of symbiotrophs and pathotrophs, but few or no ectomycorrhizal or ericoid mycorrhizal fungi were detected.

Section 3.3, Microbial Functional Traits

For this section, can the author please provide the percentage of ASV that were used in the analysis. All unknowns would be excluded, and if any one sites dataset contains a large number of unknown, then the confidence in more of less functional traits per site would be questionable. due to potentially comparing 60% of ASV in one site vs 30% of the ASV in another site (just an example). This is why it is important to state what precentage is being used in each site for this analysis.

I see the author put this below the table, but why such low numbers when there are 380 bacterial/Archaea and 257 fungal ASVs. So few are being used in the analysis (was this only for the top 40?).

Response: We have added a new paragraph to this section (lines 112-122):

Of the 151 Ascomycota ASVs queried through FUNGuild, 59 taxa (39.1%) were assigned a functional trait, with most unassigned ASVs missing family, genus and species classification. It is likely that the naming conventions from the SILVA database (e.g. Cladosporium complex) were not directly matched within FUNGuild, and some others may be missing from the dataset (Furcaspora). Within the prokaryote dataset, there were 379 ASVs queried, and 59 were assigned a functional group, most classified to at least family. Unassigned taxa were those that were missing lower rank names, missing from the database, or remained unmatched due to SILVA naming (e.g., Candidatus Kroibacter, genus RB41, and others). There were a larger proportion of named prokaryotes (down to family or genus) that were not found in the FAPROTAX database (100 ASVs, 26.4%) than Ascomycota not found in FUNGuild (9 ASVs, 6.0%).

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors! I with interest read your manuscript (сommunication) entitled: “Small farm holder cropping systems influence microbial profiles in an equatorial rainforest agroecosystem”. The topic of article is relevant and fits to Agronomy journal scope, also the article is suitable for consideration in other MDPI journals like: Land, Plants, Soil systems, Sustainability, Microbiology Research, etc. I have the following recommendations and suggestions:

L. 13 “Microbial richness and diversity tended to increase with cultivation intensity”, really increase? Probably decrease

L. 41. Please provide a short climatic description (precipitations, temperature, etc).

L. 42. You could provide a citation related to soil WRB classification (IUSS Working Group WRB. World Reference Base for Soil Resources 2014, update 2015. International soil classification system for naming soils and creating legends for soil maps. In World Soil Resources Reports; FAO: Rome, Italy, 2015; p. 182.)

LL. 74-76. Probably the description of abbreviations could be omitted because you already mentioned about it earlier.

Table 1. In addition to the statistical analyses, the result of physicochemical properties of soil, could be presented in supplementary material.

 

In general, Authors could expand the all parts of manuscript (add sentences in Introduction, Discussion, etc) but since the manuscript is presented in the form of a communication, they could provide next publication as full article.

Author Response

Dear Authors! I with interest read your manuscript (сommunication) entitled: “Small farm holder cropping systems influence microbial profiles in an equatorial rainforest agroecosystem”. The topic of article is relevant and fits to Agronomy journal scope, also the article is suitable for consideration in other MDPI journals like: Land, Plants, Soil systems, Sustainability, Microbiology Research, etc. I have the following recommendations and suggestions:

L. 13 “Microbial richness and diversity tended to increase with cultivation intensity”, really increase? Probably decrease

Response: This statement comes directly from Table 2, where both richness and diversity were greater (for both prokaryotes and Ascomycota) in sugarcane than maize-beans, and in maize-beans than forest.

L. 41. Please provide a short climatic description (precipitations, temperature, etc).

Response:  We have added the following text (lines 51-52):

Average annual rainfall in 2016 was 1512 mm with average daytime temperatures around 26 °C [citation added].

L. 42. You could provide a citation related to soil WRB classification (IUSS Working Group WRB. World Reference Base for Soil Resources 2014, update 2015. International soil classification system for naming soils and creating legends for soil maps. In World Soil Resources Reports; FAO: Rome, Italy, 2015; p. 182.)

Response: Thank you, we have added a citation to the WRB classification 4th edition (2022) to line 50.

L. 74-76. Probably the description of abbreviations could be omitted because you already mentioned about it earlier.

Response:  We have included the description of the abbreviations in the figure and table captions as they are intended to be stand-alone, as the need arises.

Table 1. In addition to the statistical analyses, the result of physicochemical properties of soil, could be presented in supplementary material.

Response:  Thank you for the suggestion. We have added this information in a new table in supplementary material (Table S1), please see lines 84-86:

In contrast, the two cropping systems occurred in a broad range of soil conditions—sandy to clay/silt rich, low to high acidity, low to high organic carbon and nitrogen (both N (%) and TN (Mg ha-1)) (Table 1; Table S1).

In general, Authors could expand the all parts of manuscript (add sentences in Introduction, Discussion, etc) but since the manuscript is presented in the form of a communication, they could provide next publication as full article.

Response: We have expanded the text, especially in Sections 2 and 3.3. We do agree that future collaboration will have more information to support a full article format, please see lines 150-152:

As this was a descriptive study based on a limited number of samples, future work will benefit from increased replication and the inclusion of other cropping systems common in the region.

Reviewer 3 Report

Comments and Suggestions for Authors

Abstract

Lines 13-18, are these findings statistically significant? If so, then please mention that/provide p-values.

Line 20, please outline a take-home statement. For instance, what implications do the findings from your research have for policymakers regarding the conservation of the rainforest in that particular area?

Methods

There is no information on the fertilizer rate and application. also, when did the sampling occur? Growing season?

Line 43, Are the soil samples from the northern part of the watershed or south?

Lines 44-45, the n numbers are the number of replicates. This is not clear. There are 5 replicates indicated in line 47. Please clarify the statements.

Line 61, which statistical software was applied?

Results

Is there any statistical significance test for the species diversity indices? It should be noted that without statistical analysis it is hard to conclude if there are any differences between the groups. If statistical tests couldn't be applied due to a limited number of replicates, this should be acknowledged within the discussion section.

Table 1. Please provide the number of replicates in the caption.

 

Table 2. Please provide the standard deviation/errors of the means.

Author Response

Abstract

Lines 13-18, are these findings statistically significant? If so, then please mention that/provide p-values.

Response: Due to the limited number of replicates (see lines 59-62 and 146-147), none of the analyses except for soil chemistry have p-values associated with them, so we cannot state p-values in the abstract, only trends. We did modify the first sentence of the third paragraph in Materials and Methods (lines 64-65) to make clear that sequence data came from ONE sample per cropping system for each location:

One 50 g soil sample was pooled for each cropping system within each location and analyzed for microbial community profiles.

Line 20, please outline a take-home statement. For instance, what implications do the findings from your research have for policymakers regarding the conservation of the rainforest in that particular area?

Response: Because this is a preliminary study that shows trends and differences in these agricultural systems, we do not feel that it would be appropriate to make any general recommendations in the abstract. We do note, in Conclusions (lines 154-157) that:

Extractive cropping practices have been shown to have the potential to reduce original SOC by 60 to 80% [3] and fundamentally alter soil chemistry and microbial communities, and their energy and nutrient cycling processes in complex ways.

Methods

There is no information on the fertilizer rate and application. also, when did the sampling occur? Growing season?

Response: We do not have specific information about the fertilizer rates or applications in this area or on the locations at which we sampled.  But we have revised and added to the following text to provide more information on sampling dates and whether crops were in session at the time of sampling (lines 52-28):

On 8 and 22 November and 6 December of 2016, we sampled (1) undisturbed soils (F) in the Kakamega Forest Reserve near Maghaka (Ma), and (2) soils in croplands in Bu-khaywa (Bu), Ikolomani (Ik), and Township (Tw) where maize-bean intercrop (MB) or sugarcane crops (Sc) had been planted continuously for at least five years. Cropping at the sample locations was in small scale farms where MB is typically done in two annual cycles during both long rain (April to June) and short rain (October to December) periods—MB and Sc crops were in session during sampling.

Line 43, Are the soil samples from the northern part of the watershed or south?

Response: North and west parts of the watershed.

Lines 44-45, the n numbers are the number of replicates. This is not clear. There are 5 replicates indicated in line 47. Please clarify the statements.

Response: We apologize for the confusion.  There were 5 replicates of soil samples for each location, as indicated at the beginning of line 59.  The n values in the first paragraph of Materials and Methods referred to the number of locations per cropping system, but we have revised the text in lines 52-61 to make the numbers clearer:

On 8 and 22 November and 6 December of 2016, we sampled (1) undisturbed soils (F) in the Kakamega Forest Reserve near Maghaka (Ma), and (2) soils in croplands in Bu-khaywa (Bu), Ikolomani (Ik), and Township (Tw) where maize-bean intercrop (MB) or sugarcane crops (Sc) had been planted continuously for at least five years. Cropping at the sample locations was in small scale farms where MB is typically done in two annual cycles during both long rain (April to June) and short rain (October to December) periods—MB and Sc crops were in session during sampling.

Five replicates of approximately 400 g from the top 20 cm of soil were collected and then pooled from each location (Ma, Bu, Ik, Tw) for each cropping system (F, n = 3; MB, n = 9; Sc, n = 9) and analyzed …

Line 61, which statistical software was applied?

Response: We have added this information to lines 72-74:

Kruskal-Wallis one-way analysis of variance on ranks and Dunn’s pairwise (p < 0.05) comparisons were used to compare soil properties among locations (Ma, Bu, Ik, Tw) and among cropping systems (F, MB, Sc) using rstatix [citation added] in R v.4.1.1.

Results

Is there any statistical significance test for the species diversity indices? It should be noted that without statistical analysis it is hard to conclude if there are any differences between the groups. If statistical tests couldn't be applied due to a limited number of replicates, this should be acknowledged within the discussion section.

Response: We acknowledged the limitations because of replicates in lines 146-149:

There were a limited number of replicates of soil community samples, but we found that cropping systems may influence the composition and functions of microbial communities relative to that of the remnant original forest.

Table 1. Please provide the number of replicates in the caption.

Response: The number of replicates (n = 5) has been added to the caption of Table 1 on line 87.

Table 2. Please provide the standard deviation/errors of the means.

Response: Because of the limitations of replicate number (n=1), we do not have standard deviations or error data available for this table.